CN107022029B

CN107022029B - Monoclonal antibody for detecting alpha-fetoprotein with high specificity and high sensitivity, kit and application

Info

Publication number: CN107022029B
Application number: CN201710179442.0A
Authority: CN
Inventors: 向军俭; 黄建芳; 孙一帆
Original assignee: Jinan University
Current assignee: Guangzhou Purui Biotechnology Co ltd
Priority date: 2017-03-23
Filing date: 2017-03-23
Publication date: 2021-03-23
Anticipated expiration: 2037-03-23
Also published as: CN107022029A

Abstract

The invention discloses a monoclonal antibody for detecting alpha fetoprotein with high specificity and high sensitivity, a kit and application. The 3 monoclonal antibodies Ab1-Ab 3 can specifically identify humanized natural hAFP in human liver cancer cells and liver cancer tissues, and have good specificity. When the antibody pairing combination Ab1+ Ab3/HRP-Ab2 is used for carrying out double-antibody sandwich ELISA detection on a sample, the linear detection range is 5-250 ng/ml, the lowest detection limit is 2ng/ml, the upper detection limit is 400ng/ml, and the linear detection range is 5-200 ng/ml and the lowest detection limit is 5ng/ml which are superior to those of an imported kit; the clinical sample detection result shows that the positive serum detection accuracy of the detection method is 100 percent, and the negative serum accuracy is 100 percent. The double-antibody sandwich ELISA method established by the invention has a larger linear range and better popularization and application values.

Description

Monoclonal antibody for detecting alpha-fetoprotein with high specificity and high sensitivity, kit and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a group of monoclonal antibodies which are high in affinity and specificity and can be combined with different epitopes of human alpha-fetoprotein, and application thereof.

Background

Human alpha fetoprotein (hAFP) is an embryonic glycoprotein synthesized from fetal liver cells and yolk sac, and has a function similar to that of Human Serum Albumin (HSA), and can be regarded as an HSA substitute during fetal development. Normally, hAFP is only present in fetal serum, and the hAFP concentration can be reduced back to normal level (<5ng/ml) after one year of fetal birth, and the concentration in normal human serum is generally not higher than 20 ng/ml. When a body has primary liver cancer, a liver cancer cell can secrete a large amount of hAFP in peripheral blood, so that the concentration of the hAFP in the blood is abnormally increased.

hAFP is one of the earliest discovered tumor markers, has long been used as an important serum molecular marker of liver cancer, fetal deficiency and embryo-related cancer, and is an important evaluation index molecule in blood test. Besides being applied to prenatal diagnosis and hepatocellular carcinoma diagnosis, the hAFP blood detection has important significance for diagnosis and disease condition monitoring of diseases such as liver injury, liver cirrhosis, hepatitis, gastrointestinal cancer, neural tube injury, endoblastoma, reproductive system cancer and the like.

China is a big-mouth individual, and prenatal diagnosis is closely related to every family; meanwhile, China is also a region with high incidence of liver cancer, the number of people dying from liver cancer in each year accounts for more than 40 percent of the world, and because liver cancer of middle and late stages basically has no effective treatment method, the national general survey and early diagnosis of the blood concentration of the primary liver cancer specific tumor antigen hAFP are the most effective measures for solving the problem of high mortality of liver cancer. Therefore, the method for detecting the serum hAFP, which is sensitive, specific, accurate, simple and convenient and has low cost, meets the urgent need of the society.

Human alpha-fetoprotein (hAFP) is used as the most specific tumor marker and prenatal diagnosis and monitoring molecule of primary liver cancer, and the monoclonal antibody thereof has extremely important practical application significance. Although anti-hAFP monoclonal antibodies have been successfully prepared at home and abroad, most of them have no clinical application value and cannot be used for reagent kit development and production. In the currently sold hAFP detection ELISA kit products at home and abroad, the linear detection range is within 20-400ng/ml, wherein the overall quality of the imported kit is obviously superior to that of the domestic products, but the linear range and the sensitivity of the imported kit still need to be further improved. In addition, because the components in serum are complex and easy to generate false positive, the change range of the hAFP concentration is extremely large (5ng/ml-10mg/ml), so that a double-antibody sandwich ELISA (DAS-ELISA) two-step method is adopted in practical detection application to improve the detection accuracy and stability, the method requires that a capture antibody and a detection antibody used for pairing have high affinity and good specificity, and the farther the spatial position distance is, the better the pairing effect is when an antibody 1-antigen-antibody 2 complex is formed, so that the continuous development of the double-antibody sandwich pairing antibody with higher sensitivity and better linear detection range is the effort direction of kit development and is also the requirement of clinical detection.

Disclosure of Invention

The technical problem to be solved by the invention is to provide monoclonal antibodies capable of specifically binding different epitopes of human alpha-fetoprotein and establish a double-antibody sandwich Elisa method to detect the alpha-fetoprotein in human serum.

The invention aims to provide a group of mouse monoclonal antibodies targeting different epitopes of human alpha fetoprotein (hAFP), and the antibodies are required to be capable of being combined with the human alpha fetoprotein with high affinity and high specificity and forming a double-antibody sandwich mode, so that the human alpha fetoprotein can be quantitatively detected.

The invention aims to provide a monoclonal antibody for detecting alpha fetoprotein with high specificity and high sensitivity.

Another purpose of the invention is to provide a kit for detecting alpha-fetoprotein with high specificity and high sensitivity.

The technical scheme adopted by the invention is as follows:

a monoclonal antibody which binds to human alpha-fetoprotein with high specificity and high affinity is Ab1, Ab2 or Ab3, and the variable region genes of the monoclonal antibody Ab1, Ab2 or Ab3 comprise a heavy chain variable region and a light chain variable region;

the amino acid sequence of the heavy chain variable region of the monoclonal antibody Ab1 is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8;

the amino acid sequence of the heavy chain variable region of the monoclonal antibody Ab2 is shown as SEQ ID NO.9, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 10;

the amino acid sequence of the heavy chain variable region of the monoclonal antibody Ab3 is shown in SEQ ID NO.11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 12.

Further, the gene sequence of the heavy chain variable region of the monoclonal antibody Ab1 is shown in SEQ ID No.1, and the gene sequence of the light chain variable region is shown in SEQ ID No. 2;

the gene sequence of the heavy chain variable region of the monoclonal antibody Ab2 is shown as SEQ ID NO.3, and the gene sequence of the light chain variable region is shown as SEQ ID NO. 4;

the gene sequence of the heavy chain variable region of the monoclonal antibody Ab3 is shown as SEQ ID NO.5, and the gene sequence of the light chain variable region is shown as SEQ ID NO. 6.

A kit for detecting human alpha-fetoprotein, comprising the monoclonal antibody of any one of the above.

A double-antibody sandwich ELISA kit for detecting human alpha-fetoprotein, which comprises any one of the monoclonal antibodies Ab1 and Ab2, or Ab3 and Ab2, or Ab1, Ab3 and Ab 2.

Further, the monoclonal antibody Ab1 or/and Ab3 is a capture antibody, and the monoclonal antibody Ab2 is a detection antibody.

A double-antibody sandwich ELISA method for detecting human alpha-fetoprotein, which uses any one of the monoclonal antibodies Ab1 or/and Ab3 as a capture antibody, and uses any one of the monoclonal antibodies Ab2 as a detection antibody; the method is useful for diagnosis or treatment of non-disease.

Further, the method specifically comprises the following steps:

s1, coating: coating the coated plate with a solution containing monoclonal antibodies Ab1 or/and Ab3 overnight;

s2, sealing: washing the coating plate coated with Ab1 or/and Ab3 with PBST, and blocking with skimmed milk powder or BSA;

s3, adding an antigen: washing the sealed coating plate by PBST, adding a plasma sample to be detected, and incubating for 50-70 min;

s4, adding a secondary antibody: washing the coated plate after the sample incubation by using PBST, adding a secondary antibody HRP-Ab2, and incubating for 30-50 min;

s5, washing the substrate by PBST, beating the substrate to be dry, adding TMB color development liquid, incubating the substrate at room temperature in a dark place, stopping color development, and reading OD by an enzyme-linked immunosorbent assay (OD)₄₅₀The value is obtained.

The monoclonal antibody of any one of the above in the preparation of liver cancer early diagnosis kits or reagents.

The monoclonal antibody of any one of the above in the preparation of a kit or a reagent for detecting human alpha-fetoprotein.

The invention has the beneficial effects that:

1) the invention discloses a mouse-derived monoclonal antibody capable of specifically binding to human alpha-fetoprotein, which can bind to different epitopes of the human alpha-fetoprotein to form a double-antibody sandwich mode, realize the quantitative detection of the alpha-fetoprotein in human serum, and has certain effects on prenatal diagnosis and early liver cancer diagnosis.

2) The 3 monoclonal antibodies Ab1-Ab 3 can specifically identify human natural hAFP in human liver cancer cells and liver cancer tissues, and Ab1-Ab 3 have no obvious reaction with cross protein HSA and have good specificity. The double-antibody sandwich ELISA method established by the group of antibodies can be used for detecting human alpha-fetoprotein, carrying out prenatal diagnosis and early diagnosis of liver cancer, and has certain commercial application value.

3) The detection sensitivity of 2 pairs of paired antibody combinations of Ab1/HRP-Ab 2 and Ab3/HRP-Ab2 is 5ng/ml, the linear detection range is 10-200ng/ml, and the detection upper line is 400 ng/ml. The linear detection range of the Ab1+ Ab3/HRP-Ab2 pairing combination is about 5-250 ng/ml, the lower detection limit can reach 2ng/ml, and the upper detection limit is 400 ng/ml.

4) The antibodies Ab1, Ab3 and Ab2 have good detection effect on hAFP, and particularly when the antibody pairing combination Ab1+ Ab3/HRP-Ab2 is used for carrying out double-antibody sandwich ELISA detection on a sample, the linear detection range is 5-250 ng/ml, the lowest detection limit is 2ng/ml, the upper detection limit is 400ng/ml, and the linear detection range is 5-200 ng/ml and the lowest detection limit is 5ng/ml which are superior to those of an imported Kit (ABCam ELISA Kit); the clinical sample detection result shows that the positive serum detection accuracy of the detection method is 100 percent, and the negative serum accuracy is 100 percent. The double-antibody sandwich ELISA method established by the invention has a larger linear range and better popularization and application values.

Drawings

FIG. 1 is a relative affinity assay for monoclonal antibodies Ab1 to Ab 4;

FIG. 2 shows that monoclonal antibody Ab1-Ab 4 performs immunofluorescence staining detection on HepG2 human hepatoma cells;

FIG. 3 shows the immunohistochemical detection of monoclonal antibody Ab1-Ab 4 on human primary liver cancer tissues;

FIG. 4 shows the 3 CDR (complementarity determining region) regions in the heavy chain variable region of Ab1 antibody;

FIG. 5 shows the 3 CDR (complementarity determining region) regions in the light chain variable region of Ab1 antibody;

FIG. 6 shows the 3 CDR (complementarity determining region) regions in the heavy chain variable region of Ab2 antibody;

FIG. 7 shows the 3 CDR (complementarity determining region) regions in the light chain variable region of Ab2 antibody;

FIG. 8 shows the 3 CDR (complementarity determining region) regions in the heavy chain variable region of Ab3 antibody;

FIG. 9 shows the 3 CDR (complementarity determining region) regions of the light chain variable region of Ab3 antibody;

FIG. 10 is a detection curve of a double antibody sandwich ELISA for different partner antibodies;

FIG. 11 is a linear standard curve of paired antibody Ab1/HRP-Ab 2 double antibody sandwich ELISA detection;

FIG. 12 is a linear standard curve of paired antibody Ab3/HRP-Ab2 double antibody sandwich ELISA detection;

FIG. 13 is a linear standard curve for paired antibody Ab1+ Ab3/HRP-Ab2 double antibody sandwich ELISA detection.

Detailed Description

The present invention will be further described with reference to the following examples, but is not limited thereto.

EXAMPLE 1 preparation of monoclonal antibodies

Antigen Human alpha-fetoprotein (Human Myoglobin) was purchased from Guilin England Biotechnology Ltd, isolated and purified from Human umbilical cord blood at a concentration of 7,6mg/ml, is a natural Human alpha-fetoprotein molecule with a molecular weight of 69kDa and consists of 609 amino acid residues. hAFP is a multifunctional transporter with high expression in fetal period and silent postnatal expression. The concentration of hAFP in serum is a direct or indirect evaluation index of various diseases, and is particularly commonly used for prenatal diagnosis, liver cancer diagnosis and disease condition monitoring.

The preparation method of the anti-human alpha-fetoprotein murine monoclonal antibody comprises the following steps:

(1) animal immunization

1) Primary immunization: fully emulsifying 30 mu g of antigen with equivalent volume of Freund's complete adjuvant, and performing subcutaneous multi-point injection on the mouse;

2) and (3) secondary immunization: after 2 weeks, the antigen amount same as that of the primary immunization is added with the same amount of Freund incomplete adjuvant, and the mixture is fully emulsified and then injected into multiple subcutaneous points;

3) three times of immunization: after 2 weeks, the antigen amount same as that of the primary immunization is fully emulsified by adding equivalent Freund incomplete adjuvant and then is injected into multiple subcutaneous points (after 10 days, tail vein blood collection is carried out to measure the titer);

4) and (3) boosting immunity: after the third immunization titer meets the cell fusion requirement, the same antigen amount of the primary immunization is used for intraperitoneal injection without adjuvant;

5) and taking spleens for fusion after 72 h.

(2) Cell fusion

1) Preparation of feeder cells: taking a healthy Balb/c mouse, picking eyeballs, collecting blood, killing neck dislocation after blood is completely discharged, sterilizing and fixing body surface, cutting skin from thigh, exposing peritoneum, and sterilizing peritoneum with alcohol cotton ball. Injecting 10mL RPMI1640 basic Medium (purchased from RPMI Medium 1640basic, gibco, cat # 8114056, adding penicillin and streptomycin before use in an amount of 1mL penicillin and streptomycin double antibody containing 100U per 100mL Medium) into abdominal cavity with a 5mL syringe, fixing the syringe with the right hand, gently massaging the abdomen with alcohol cotton ball held by the left hand, withdrawing the liquid in the abdominal cavity, injecting into a prepared sterile 10mL centrifuge tube, centrifuging for 7 minutes at 1000rmp, resuspending with 10% fetal bovine serum RPMI1640 (containing HAT) complete Medium, diluting to about 2 × 10⁵One/ml, then added to a 96-well plate at 100. mu.l per well, and placed in a cell incubator (37 ℃, 5% CO)₂) And (4) preparing for later use.

2) Taking a mouse myeloma cell SP2/0 with logarithmic growth, washing the mouse myeloma cell SP2/0 with an RPMI1640 basic culture medium, and counting the cells after blowing, suspending and diluting;

3) washing and grinding a mouse spleen and an RPMI1640 basic culture medium to prepare a single spleen cell suspension, and counting;

4) myeloma cells and splenocytes were mixed in a 1:10, and centrifuging at 1000rpm for 7 min;

5) discarding the supernatant, completely sucking the residual liquid by using a dropper, dropwise adding 1mL of polyethylene glycol (PEG) within 1min under the condition of 37 ℃ water bath, standing for 90 seconds, and dropwise adding 15mL of RPMI1640 basic culture medium within 2-4 min to terminate the reaction;

6) centrifuging at 1000rpm for 7min, discarding the supernatant, gently diluting with 100mL 10% fetal bovine serum RPMI1640 (containing HAT)Suspending; dripping into 96-well plate with feeder cells, 100 μ l/well; 37 ℃ and 5% CO₂Culturing in an incubator.

(3) Selection and cloning of fusion cells

1) Taking cell culture supernatant about 7 days after cell fusion, performing indirect ELISA detection by using an ELISA plate coated with 30 ng/hole of human alpha-fetoprotein, and screening positive holes; sera taken before the fusion of the immunized mice were used as positive control and SP2/0 supernatant was used as negative control. Finally, 14 positive cell strains are screened.

2) After 3-4 continuous cloning experiments are carried out on the 14 cell strains, the titer of the supernatant of the 3 cell strains is found to be unstable and is not satisfactory. Finally, 12 positive hybridoma cell strains (named AB1-AB 12) capable of stably secreting anti-hAFP monoclonal antibodies are obtained through cloning and co-screening, and can basically meet the requirements of further antibody pairing experiments.

(4) Preparation of ascites type monoclonal antibody

1) Taking a female Balb/c mouse aged 10 weeks, and injecting 0.5mL Freund's incomplete adjuvant into the abdominal cavity;

2) mice were inoculated intraperitoneally 1 week later with about 5 x 10⁵2, inoculating the hybridoma cells for 7-12 days to induce ascites;

3) extracting ascites when the ascites is as much as possible;

4) after ascites regeneration and accumulation, re-pumping by the same method at intervals of 1-2 days, centrifuging the ascites at 3000rpm for 10min after pumping, and taking the supernatant and storing at-20 ℃.

(5) Purification and potency detection of monoclonal antibodies

1) Centrifuging ascites at 4 deg.C and 12000rpm for 30min, and collecting supernatant;

2) dropwise adding an equal volume of saturated ammonium sulfate solution under continuous stirring, and standing overnight at 4 ℃;

3) centrifuging the overnight liquid at 7500rpm at 4 deg.C for 30min, discarding the supernatant, and re-dissolving the precipitate with 0.1M PBS;

4) desalting the composite solution by using a desalting column, wherein the specific operation steps are as follows:

firstly, column balancing: using 0.1M PBS (5-10 times of the volume of the column), balancing the column, flushing out 20% ethanol in the column, and zeroing the nucleic acid protein instrument;

sample loading: before sample loading, the constant flow pump is closed, then sample loading is carried out slowly, 0.1M PBS is continuously introduced after sample loading is finished, when the value A begins to rise, liquid (target protein) is collected, and when the value A falls below 10, collection is stopped. The collected sample was continued for further purification.

③ balancing the columns: when the value of A is "0", passing through the column with 0.1M PBS (5 times the column volume);

and fourthly, preservation: the column was kept under 20% ethanol (5 column volumes).

5) And purifying the desalted Protein by using a Protein G affinity chromatography column.

The purification steps are as follows:

firstly, column balance column installation: using 0.1M PBS (5-10 times of the volume of the column), balancing the column, flushing out 20% ethanol in the column, and zeroing the nucleic acid protein instrument;

sample loading: before loading, the constant flow pump is closed, then the sample is loaded slowly, when the value A begins to rise, liquid (hetero protein) is collected to prevent the protein from being unbound to the column, and when the sample is loaded, 0.1M PBS is added for dilution, so that the protein is almost completely loaded to the column;

③ elution: eluting target protein with eluent when A value is reduced to "0", collecting liquid when A value begins to rise (protein is negatively charged and weakly alkaline, and adding a certain amount of neutralizing liquid into the collecting tube to maintain pH of the collected liquid above 7.0);

fourthly, column balancing: when the value of A is "0", passing through the column with 0.1M PBS (5 times the column volume);

preservation: the column was kept under 20% ethanol (5 column volumes).

Sixthly, after the protein is ultrafiltered and concentrated, a small amount of the protein is taken to carry out SDS-PAGE gel electrophoresis to identify the purity, and the antibody titer is detected by adopting an ELISA method.

The 12 hybridoma cell strains capable of stably secreting the anti-hAFP monoclonal antibody are successfully screened, wherein 5 monoclonal antibodies (Ab 1-Ab 5) secreted by the strains have extremely high ascites titer and can reach 3000 ten thousand.

(6) Relative affinity detection of monoclonal antibodies

Respectively diluting the ascites or supernatant of the 12 monoclonal antibodies screened out to IgG antibody concentration of 10 according to the IgG antibody concentration in the ascites⁵ng/ml, 10-fold gradient dilution and plotting (see FIG. 1).

When the antibody dilution curve reached OD₄₅₀Half the maximum, the lower the corresponding IgG antibody concentration (P50 value, 50% of maximum binding), the higher the corresponding affinity. As shown in FIG. 1, among the 12 monoclonal antibodies prepared, 4 antibodies (Ab 1-Ab 4) have higher or close affinity to that of the imported standard antibodies AFP-01 (labeled with an arrow in the figure) and AFP-11 (labeled with an arrow in the figure 11), which indicates that the 5 antibodies have extremely high affinity and are the potential optimal selection of the paired antibodies.

Through the relative affinity determination, 12 hybridoma cell strains capable of stably secreting the anti-hAFP monoclonal antibody are successfully screened, wherein the affinity of 4 monoclonal antibodies (Ab 1-Ab 4) secreted by the 4 monoclonal antibodies is higher than or similar to that of ABCam paired antibodies AFP-01 and AFP-11, so that the 4 monoclonal antibodies all meet the affinity requirement of commercial paired antibodies.

Example 2 analysis of the specificity of monoclonal antibodies

The 4 selected important monoclonal antibodies Ab1-A b4 have very high affinity to the immunogen hAFP, and in order to further identify the specificity and verify whether the monoclonal antibodies can be specifically combined with the naturally occurring hAFP, the following two experiments are designed at the cell level and the tissue level respectively.

1) HepG2 human liver cancer cell immunofluorescence staining

It is known that hAFP can be synthesized in large amount in human hepatoma cell HepG2, and cell fluorescent staining experiments were performed using 4 monoclonal antibodies Ab1-Ab 4.

The experimental method comprises the following steps: one day before experiment, HepG2 cell in good state is taken to prepare cell suspension, and cell concentration is 5X 10 by cell counting⁵Spreading the quantity of the individual/hole into a hole of a six-hole plate on which a cover glass is placed; culturing in an incubator for one day, and starting a dyeing step when the cell confluency reaches about 70%; flushing the cover glass with PBS once and abandoningAfter the liquid is exhausted, fixing the cell cells with 4% paraformaldehyde for 10min, and then washing with PBS for 3 times and 5 min/time; preparing a 3% triton-X100 solution by using PBS, carrying out membrane permeabilization on the cells, treating for 10min, washing for 3 times by using PBS, and washing for 5 min/time; 5% skimmed milk powder is sealed for 30min, and washed for 3 times by PBS; sixthly, adding a properly diluted monoclonal antibody on a cover glass, taking an irrelevant antibody as a negative control group, incubating for 1h at 37 ℃, and washing for 3 times by PBS; seventhly, adding FITC-labeled donkey anti-mouse IgG secondary antibody diluted by 1:100 times, incubating for 40min at 37 ℃, and washing for 2 times by PBS; adding 0.5ug/ml DAPI staining solution to dye for 10min, and washing with PBS for 3 times; ninthly, 20ul of sealing agent is added for sealing, observation is carried out under a confocal microscope (400 times), and the result is stored.

The experimental results are as follows: the observation results show (FIG. 2, magnification: 400 ×), in the experimental group corresponding to 4 monoclonal antibodies, the HepG2 cells all showed obvious green fluorescence, while the irrelevant antibody control group cells did not observe green fluorescence, which indicates that Ab1-Ab 4 monoclonal antibodies can specifically recognize hAFP molecules in the cytoplasm of human hepatoma cells, and proves that the 4 monoclonal antibodies can specifically recognize naturally-occurring human AFP molecules.

2) Immunohistochemistry of human liver cancer tissue:

when a human body has primary liver cancer, liver cancer cells can secrete a large amount of AFP in peripheral blood. Immunohistochemical analysis was performed on liver tissues from patients with primary liver cancer.

The experimental method comprises the following steps: firstly, fixing and embedding: fixing tissue block with 4% paraformaldehyde, subjecting to 70% ethanol for 30min, 80% ethanol for 30min, 90% ethanol for 30min 2 times, 95% ethanol for 30min 2 times, 100% ethanol for 30min 2 times, xylene for 30min 2 times, and paraffin at 55 deg.C for 30min 2 times, and wax embedding tissue block in mold; cutting into slices: placing the tissue slices with the thickness of 3-5um on a polylysine activated glass slide, and standing overnight at 60 ℃; and thirdly, dewaxing and entering water: soaking the slices in xylene for 5min 2 times, 100% ethanol for 5min 2 times, 95% ethanol for 5min 2 times, 90% ethanol for 5min 2 times, 85% ethanol for 5min 2 times, 75% ethanol for 5min 2 times, and washing with PBS for 2 times; fourthly, washing with 1% methanol hydrogen peroxide for 10min at room temperature for 1 time by distilled water and washing with PBS for 3 times; fifthly, putting the slices into 0.01M citrate buffer solution (pH 6.0) for repairing, and washing for 3 times by PBS in a microwave oven for 10 min; closing: sealing with 5% skimmed milk powder at room temperature for 20min, and washing; seventhly, enzyme-labeled antibodies (Ab1-HRP, Ab2-HRP, Ab3-HRP and Ab4-HRP) which are diluted moderately are respectively dripped on the slices, and the slices are washed for 5 times by PBS after being kept overnight at 4 ℃; dripping horseradish enzyme labeled streptavidin-avidin working solution (S-A/HRP) on the slice, washing for 3 times by PBS at 37 ℃ for 20 min; ninthly, DAB color development: adding a drop of color developing agent A, B, C into 1ml of distilled water of a DAB color developing kit, uniformly mixing, adding the mixture to a specimen, developing for 6min, and terminating water washing; counter staining cell nucleus with hematoxylin in R for 1min, washing with water, differentiating with 1% hydrochloric acid alcohol, washing with 1% amine water, washing with water, dehydrating with 70% ethanol for 5min, 80% ethanol for 5min, 90% ethanol for 5min 2 times, 95% ethanol for 5min 2 times, 100% ethanol for 5min 2 times, clearing with xylene for 5min 2 times, and sealing with neutral resin.

The experimental results are as follows: the results of the examination showed that (FIG. 3, magnification: 400 ×), most of the cytoplasmic portions of the cells were stained brown in the liver cancer tissue sections corresponding to 4 monoclonal antibodies Ab1-Ab 4, indicating that these cells have the ability to secrete a large amount of hAFP, i.e., the cells are cancerous hepatocytes, and the brown cells may be non-cancerous hepatocytes. Meanwhile, the histochemical result also shows that the 4 monoclonal antibodies Ab1-Ab 4 can specifically recognize and combine hAFP molecules in the cytoplasm of hepatoma cells in the hepatoma tissues.

3) Cross reaction analysis

The main cross-reactive protein of hAFP is known as human serum albumin HSA, and the presence of cross-reaction of human serum albumin HAS in monoclonal antibodies Ab1-Ab 3 is detected by indirect ELISA method by using 40mg/ml HSA as cross-reactive antigen according to HSA concentration in human blood. The experiment set up an HSA cross-reactive group and a PBS negative control group. The experiment was repeated 3 times. (AFP 400ng/ml, HSA 40mg/ml)

The specific operating method of the indirect ELISA assay is as follows: diluting hAFP antigen with 0.05M carbonate buffer (coating solution) of pH9.6 to 500ng/ml, coating at 37 deg.C for 3h or overnight at 4 deg.C with 100 μ l/well, and washing the plate with PBS-T for 3 times and 3 min/time; 5% skimmed milk powder 200 μ l/well, sealing at 37 deg.C for 1h, washing with PBS-T for 3 times; diluting the polyclonal antiserum and the negative control serum of the mouse to be detected in a 2-fold ratio gradient multiple ratio manner, adding the diluted polyclonal antiserum and the negative control serum into a hole with the concentration of 100 ul/hole, incubating for 1h at 37 ℃, and washing for 3 times by PBS-T; adding 100ul of goat anti-mouse IgG antibody (diluted 1: 8000) labeled with HRP, incubating at 37 ℃ for 40min, and washing 5 times with PBS-T; TMB bottomDeveloping the color of the materials at 37 ℃ in dark for 10 min; 2M H₂SO₄The reaction was stopped at 50. mu.L/well and the A value was measured at a microplate reader wavelength of 450 nm.

The detection result shows that the monoclonal antibody Ab1 HAS weak cross reaction with high-concentration HAS (the high concentration is 40mg/ml), and Ab2 and Ab3 have no cross reaction with HSA.

The results of the specific identification of the monoclonal antibodies show that 3 monoclonal antibodies Ab1 to Ab3 can specifically identify natural hAFP of human origin in human liver cancer cells and liver cancer tissues, Ab2 and Ab3 have no cross reaction with high-concentration cross protein HSA, and Ab1 has weak cross reaction with high-concentration HSA.

Example 3 sequence and structural analysis of monoclonal antibodies Ab1-Ab 3

Further sequence and structural analyses of the obtained monoclonal antibodies Ab1 to Ab3 revealed that the variable regions of Ab1 to Ab3 all included heavy chain variable regions and light chain variable regions.

(1) From the gene sequence level:

the gene sequence of the heavy chain variable region of the monoclonal antibody Ab1 is shown as SEQ ID NO.1, and the gene sequence of the light chain variable region is shown as SEQ ID NO. 2;

the gene sequence of the heavy chain variable region of the monoclonal antibody Ab3 is shown in SEQ ID NO.5, and the gene sequence of the light chain variable region is shown in SEQ ID NO. 6.

(2) From the polypeptide sequence level:

the amino acid sequence of the heavy chain variable region of monoclonal antibody Ab1 is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8.

The amino acid sequence of the heavy chain variable region of monoclonal antibody Ab2 is shown in SEQ ID NO.9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 10.

The amino acid sequence of the heavy chain variable region of monoclonal antibody Ab3 is shown in SEQ ID NO.11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 12.

(3) Structural features of the variable regions

The Ab1 heavy chain variable region consists of 360 bases and encodes 119 amino acids, and the variable region contains 3 CDR (complementarity determining region) regions. CDR1 encodes 5 amino acids, CDR2 encodes 17 amino acids, and CDR3 encodes 11 amino acids (as shown in fig. 4). The homology between the framework region of the variable region and other murine antibodies is higher than 91%, and the 3 CDR regions are specific sequences and have differences with the heavy chain variable region CDR regions of other murine antibodies.

The variable region of monoclonal antibody Ab1 light chain consists of 314 bases and encodes 104 amino acids, and the variable region contains 3 CDR (complementarity determining region) regions. CDR1 encodes 11 amino acids, CDR2 encodes 7 amino acids, and CDR3 encodes 9 amino acids (as shown in fig. 5). The homology between the framework region of the variable region and other murine antibodies is higher than 97.1%, and 3 CDR regions are specific sequences, which are different from the CDR regions of the variable region of other murine antibodies.

The heavy chain variable region of monoclonal antibody Ab2 consists of 354 bases and encodes 118 amino acids, and the variable region contains 3 CDR (complementary determinant) regions. CDR1 encodes 5 amino acids, CDR2 encodes 17 amino acids, and CDR3 encodes 10 amino acids (as shown in fig. 6). The homology between the framework region of the variable region and other murine antibodies is higher than 94.4%, and the 3 CDR regions are specific sequences, which are different from the heavy chain variable region CDR regions of other murine antibodies.

The variable region of monoclonal antibody Ab2 light chain consists of 313 bases and encodes 104 amino acids, and the variable region contains 3 CDR (complementarity determining region) regions. CDR1 encodes 11 amino acids, CDR2 encodes 7 amino acids, and CDR3 encodes 9 amino acids (as shown in fig. 7). The homology of the framework region of the variable region with other murine antibodies is more than 94.2%, and 3 CDR regions are specific sequences, which are different from the CDR regions of the variable region of other murine antibodies.

The heavy chain variable region of monoclonal antibody Ab3 consists of 357 bases and encodes 119 amino acids, and the variable region contains 3 CDR regions. CDR1 encodes 5 amino acids, CDR2 encodes 17 amino acids, and CDR3 encodes 11 amino acids (as shown in fig. 8). The homology between the framework region of the variable region and other murine antibodies is higher than 94.8%, and the 3 CDR regions are specific sequences, which are different from the heavy chain variable region CDR regions of other murine antibodies.

The variable region of monoclonal antibody Ab3 light chain consists of 314 bases and encodes 104 amino acids, and the variable region contains 3 CDR (complementarity determining region) regions. CDR1 encodes 11 amino acids, CDR2 encodes 7 amino acids, and CDR3 encodes 9 amino acids (as shown in fig. 9). The homology between the framework region of the variable region and other murine antibodies is higher than 97.1%, and 3 CDR regions are specific sequences, which are different from the CDR regions of the variable region of other murine antibodies.

The function of the 3 strains of anti-human alpha-fetoprotein antibodies (Ab 1-Ab 3) obtained by the invention is determined by specific nucleotide sequences in antibody light and heavy chain variable region antigen complementary determining groups (complementary determining regions CDRs) CDR1, CDR2 and CDR3 (functional active regions), and corresponding amino acid sequences form different epitopes on the antibody specifically binding human alpha-fetoprotein.

Example 4 antibody pairing for double antibody sandwich ELISA assays

Purified, HRP-labeled 7 monoclonal antibodies (Ab1, Ab2, Ab3, Ab4, Ab 5, Ab 6, Ab 10) were selected for antibody pairing experiments. The experiment set up an HSA cross-reactive group and a PBS negative control group. The experiment was repeated 3 times. (AFP 400ng/ml, HSA 40 mg/ml).

The double-antibody sandwich ELISA detection method comprises the following specific steps:

s1, coating: diluting the captured monoclonal antibody to a working concentration of 3-8 mu g/ml by using a carbonate buffer solution with the pH value of 9.6, adding 100 mu l of the carbonate buffer solution into each hole, and coating overnight at the temperature of 4 ℃;

s2, sealing: PBST (containing 0.05% Tween) 3 times, 3 min/time, with 5% skimmed milk powder or 2% BSA for blocking, 200 μ l per well, 37 ℃ incubation for 1 h;

s3, adding an antigen: PBST is washed for 3 times, 3 min/time, after being patted dry, human N-terminal brain natriuretic peptide precursor antigen (10-10000 g/ml) is added in turn, 50 mu l of each hole is incubated for 1h at 37 ℃;

s4, adding a secondary antibody: PBST is washed for 3-5 times, a secondary antibody (HRP-labeled detection antibody) is diluted by 5% skimmed milk powder with 8000 times, 50 mu l of the secondary antibody is added into each hole, and the mixture is incubated for 45min at 37 ℃;

s5, washing for 3-5 times by PBST (Poly-p-phenylene benzobisoxazole) (3 min each time), and beatingAdding TMB developing solution after drying, incubating for 10-15 min at room temperature in dark place, and reading OD by an enzyme-linked immunosorbent assay (OD)₄₅₀The value is obtained.

TABLE 1 double antibody Sandwich ELISA method for screening paired antibodies

Note: the "+" number represents OD₄₅₀High and low of the value: "+">1.0，“++”>2.0，“+++”>3.0; "-" represents OD450 value<0.5; "+" indicates cross-reaction.

The results of the pairing are shown in table 1, and of the 49(7 × 7) antibody pairing combinations, 13 combinations can form a double antibody sandwich pairing, wherein the pairing effect is better, 8 pairs of antibodies are provided, and the total 4 combinations have no cross reaction with HSA and have better pairing effect: ab1/HRP-Ab 2, Ab1/HRP-Ab 4, Ab3/HRP-Ab2, Ab3/HRP-Ab 4.

Example 5 sensitivity and Linear detection Range of double antibody Sandwich ELISA detection

The experimental method comprises the following steps: ab1, Ab3 and Ab1+ Ab3 (capture antibodies Ab1 and Ab3 are mixed in a ratio of 1: 1) are respectively used as capture antibodies, HRP-Ab2 is used as a detection antibody, the detection is carried out by using a double-antibody sandwich ELISA detection method (described in example 4), corresponding detection curves and standard curves are given, and the sensitivity and the linear detection range of each double-antibody sandwich ELISA detection under different capture antibody conditions are detected.

The experimental results are as follows:

FIG. 10 is a detection curve of a double antibody sandwich ELISA for different partner antibodies; FIGS. 11-13 are linear standard curves for paired antibodies Ab1/HRP-Ab 2, Ab3/HRP-Ab2, Ab1+ Ab3/HRP-Ab2, respectively, in double-antibody sandwich ELISA detection.

As can be seen from FIGS. 10-13, the detection curves of 2 pairs of paired antibody combinations, Ab1/HRP-Ab 2 and Ab3/HRP-Ab2, are very similar, the detection sensitivities are both 5ng/ml, the linear detection range is 10-200ng/ml, and the detection upper line is 400 ng/ml.

When the same concentration of HRP-Ab2 is used as the detection antibody, when the capture antibodies Ab1 and Ab3 are coated in a 1:1 mixed mode, the sensitivity and the linear detection range of Ab1+ Ab3/HRP-Ab2 are slightly improved compared with Ab1/HRP-Ab 2 and Ab3/HRP-Ab 2; at the bottom and the top of the detection curve, the OD value of Ab1+ Ab3/HRP-Ab2 is obviously higher than that of Ab1/HRP-Ab 2 and Ab3/HRP-Ab2 (figure 10), the linear detection range of the Ab1+ Ab3/HRP-Ab2 pairing combination is about 5-250 ng/ml, the lower detection limit is 2ng/ml, and the upper detection limit is 400ng/ml (figures 10 and 13). This indicates that the mixed coating group Ab1+ Ab3/HRP-Ab2 has slightly improved antigen capturing ability and double antibody sandwich complex forming ability compared with the single coating group.

Example 6 application of the double antibody sandwich ELISA assay of the present invention to clinical sample detection

120 cases (118 cases of pregnant women and 2 cases of primary liver cancer) of hAFP positive patient sera and 40 cases of negative normal human sera are collected, and the detection is respectively carried out by using the paired antibody Ab1+ Ab3/HRP-Ab2 double-antibody sandwich ELISA and imported ABCam ELISA Kit, and the sample detection accuracy rates of the two cases are counted (Table 2).

The paired antibody Ab1+ Ab3/HRP-Ab2 double-antibody sandwich ELISA detection method comprises the following steps:

s1, coating: capture antibodies Ab1 and Ab3 were mixed at a ratio of 1:1, diluted to a working concentration of 7.5 μ g/ml with carbonate buffer ph9.6, 100 μ l per well was added to the coated plate and coated overnight at 4 ℃;

s3, adding an antigen: PBST is washed for 3 times, 3 min/time, after being patted dry, human alpha fetoprotein standard antigen and human serum sample which are diluted by two times with sample diluent with different concentrations are added, and the adding amount is 80 mul; incubating at 37 ℃ for 1 h;

s4, adding a secondary antibody: PBST was washed 3-5 times, and a secondary antibody (HRP-labeled detection antibody HRP-Ab2) was diluted 8000-fold with 5% skim milk powder, 50. mu.l per well, incubated at 37 ℃ for 1 h;

s5, washing the membrane for 5 times and 3 min/time by using PBST, adding a developing solution after drying the membrane, incubating the membrane for 10min at room temperature in a dark place, and then stopping the incubation, and reading OD by using an enzyme-linked immunosorbent assay (OD)₄₅₀The value is obtained.

Meanwhile, 120 cases (118 cases of pregnant women and 2 cases of primary liver cancer) of hAFP positive patient serum and 40 cases of negative normal human serum were tested with an imported kit (purchased from ABCam, Cat. Ltd., cat # Ab108631), and the test method was performed strictly according to the kit instructions. And recording the detection results of the two, and verifying the accuracy of sample detection.

TABLE 2 test results of samples tested by different methods

Statistical results show (Table 2), the hAFP detection method established by using the paired antibody combination Ab1+ Ab3/HRP-Ab2 and the ABCam ELISA Kit can effectively detect AFP molecules in human serum. Wherein, the detection accuracy rate of the imported kit to the negative and positive serum samples is 100 percent, and the detection is stable and accurate; the detection method disclosed by the invention is proved to be capable of well detecting the hAFP antigen in the serum.

In conclusion, the antibodies Ab1, Ab3 and Ab2 found by the invention have good detection effect on hAFP, and particularly when the antibody pairing combination Ab1+ Ab3/HRP-Ab2 is used for carrying out double-antibody sandwich ELISA detection on a sample, the linear detection range is 5-250 ng/ml, the lowest detection limit is 2ng/ml, the upper detection limit is 400ng/ml, and the linear detection range is superior to the linear range of 5-200 ng/ml and the lowest detection limit is 5ng/ml of an imported Kit (ABCam ELISA Kit); the clinical sample detection result shows that the positive serum detection accuracy of the detection method is 100 percent, and the negative serum accuracy is 100 percent. The double-antibody sandwich ELISA method established by the invention has a larger linear range and better popularization and application values. The double-antibody sandwich ELISA method established by the antibody can be used for detecting human alpha-fetoprotein, carrying out prenatal diagnosis and early diagnosis of liver cancer, and has good commercial application value.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> river-south university

<120> monoclonal antibody for detecting alpha fetoprotein with high specificity and high sensitivity, kit and application

<130>

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 360

<212> DNA

<213> Artificial sequence

<400> 1

taggtgaaac tgcagcagtc tggggcagaa cttgtgaagc caggggcctc ggtcaagttg 60

tcctgttcag cttctggctt caacattcaa gactccttca tgcactgggt gaaacagagg 120

cctgaacagg gcctggagtg gattggaggg attgatcctg caaatggaaa tattagatat 180

gacccgaaat tccaggacaa ggccactatg acatccgaca catcctccaa tacagcctac 240

ctgaccctca acagcctgac atctgaggac actgccgtct attactgtgt tagaggacaa 300

ctcggaggtc gaggctggtt tgtttactgg ggccaaggga ccacggtcac cgtctcctca 360

<210> 2

<211> 314

<212> DNA

<213> Artificial sequence

<400> 2

gacattcagc tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggaaattagt gattacttaa tctggcttca gcagaaacca 120

gatggaactt ttaaacgcct gatctacgcc gcgtccactt tagattctgg tgtcccaaaa 180

aggttcagtg gcagtaagtc tgggtcagat ttttctctca ccatcagcag ccttgagtct 240

gaagattttg cagactatta ctgtctacaa tatgctagtt ttccgtacac gttcggaggg 300

gggaccaagc tgga 314

<210> 3

<211> 354

<212> DNA

<213> Artificial sequence

<400> 3

gtcaaactgc agcagtcagg gcctgagctg gtgaggcctg gggaatcagt gaagatttcc 60

tgcaagggtt ccggctacac attcactgat tattctatgc actgggtgaa gcagagtcat 120

gcaaagagtc tagagtggat tggacttatt agtatttact atgataatac aaactacaac 180

cagaagttta agggcaaggc cacaatgact gtagacaaat cctccagcac agcctatatg 240

gaacttgcca gattgacatc tgaggattct gccatctatt actgtgcaag aagggactat 300

gattacgacg agtttgctta ctggggccaa gggaccacgg tcaccgtctc ctca 354

<210> 4

<211> 313

<212> DNA

<213> Artificial sequence

<400> 4

gacattcagc tgacccagtc tccatcctcc atgtatgcat cgctgggaga gagagtcact 60

atcacttaca aggcgagtca ggacattaaa agctatttaa gctggtgcca gcagaaacca 120

tggaaatctc ctaagaccct gatctattat tcaacaagct tggcagatgg ggtcccatca 180

agattcagtg gcagtggatc tgggcaagat tattctctaa ccatcagcag cctggagtct 240

gacgatacag cttcttatta ctgtctacac catggtgaga gccctccacg ttcggagggg 300

ggaccaagct gga 313

<210> 5

<211> 357

<212> DNA

<213> Artificial sequence

<400> 5

gtcaagctgc aggagtctgg ggcagaactt gtgaagccag gggcctcagt caagttgtcc 60

tgcacagctt ctggcttcaa cattaaagac acctatatgc actgggtgaa gcagaggcct 120

gaacagggcc tggagtggat tggagggatt gatcctgcga atggtaagac taaatttgac 180

ccgaagttcc agggcaaggc cactataaca gcagacacat cctccaacac agcctacctg 240

cacctcagcc gcctgacatc tgacgacacg gccgtctatt actgtgttag agggcaagtc 300

ggaggtcgag gctggtttgc ttactggggc caagggacca cggtcaccgt ctcctca 357

<210> 6

<211> 314

<212> DNA

<213> Artificial sequence

<400> 6

gacattcagc tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggaaattagt ggttacttaa gctggcttca gcggaaacca 120

gatggaacta ttaaacgcct gatctacgcc gcatccactt tagattctgg tgtcccaaaa 180

aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240

gaagattttg cagactatta ctgtctacag tatgctagtt atccgtacac gttcggaggg 300

gggaccaagc tgga 314

<210> 7

<211> 119

<212> PRT

<213> Artificial sequence

<400> 7

Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser

1 5 10 15

Val Lys Leu Ser Cys Ser Ala Ser Gly Phe Asn Ile Gln Asp Ser Phe

20 25 30

Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly

35 40 45

Gly Ile Asp Pro Ala Asn Gly Asn Ile Arg Tyr Asp Pro Lys Phe Gln

50 55 60

Asp Lys Ala Thr Met Thr Ser Asp Thr Ser Ser Asn Thr Ala Tyr Leu

65 70 75 80

Thr Leu Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Val

85 90 95

Arg Gly Gln Leu Gly Gly Arg Gly Trp Phe Val Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 8

<211> 104

<212> PRT

<213> Artificial sequence

<400> 8

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Asp Tyr

20 25 30

Leu Ile Trp Leu Gln Gln Lys Pro Asp Gly Thr Phe Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Lys Ser Gly Ser Asp Phe Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu

100

<210> 9

<211> 118

<212> PRT

<213> Artificial sequence

<400> 9

Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Glu Ser

1 5 10 15

Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr Ser

20 25 30

Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile Gly

35 40 45

Leu Ile Ser Ile Tyr Tyr Asp Asn Thr Asn Tyr Asn Gln Lys Phe Lys

50 55 60

Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met

65 70 75 80

Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Arg Asp Tyr Asp Tyr Asp Glu Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 10

<211> 104

<212> PRT

<213> Artificial sequence

<400> 10

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Ile Thr Tyr Lys Ala Ser Gln Asp Ile Lys Ser Tyr

20 25 30

Leu Ser Trp Cys Gln Gln Lys Pro Trp Lys Ser Pro Lys Thr Leu Ile

35 40 45

Tyr Tyr Ser Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Asp Asp Thr Ala Ser Tyr Tyr Cys Leu His His Gly Glu Ser Pro Pro

85 90 95

Arg Ser Glu Gly Gly Pro Ser Trp

100

<210> 11

<211> 119

<212> PRT

<213> Artificial sequence

<400> 11

Val Lys Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser

1 5 10 15

Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr

20 25 30

Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly

35 40 45

Gly Ile Asp Pro Ala Asn Gly Lys Thr Lys Phe Asp Pro Lys Phe Gln

50 55 60

Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu

65 70 75 80

His Leu Ser Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys Val

85 90 95

Arg Gly Gln Val Gly Gly Arg Gly Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 12

<211> 104

<212> PRT

<213> Artificial sequence

<400> 12

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Arg Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu

100

Claims

1. A monoclonal antibody which binds to human alpha-fetoprotein with high specificity and high affinity is Ab2, and the variable region gene of the monoclonal antibody Ab2 comprises a heavy chain variable region and a light chain variable region;

the amino acid sequence of the heavy chain variable region of the monoclonal antibody Ab2 is shown as SEQ ID NO.9, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 10.

2. A double-antibody sandwich ELISA kit for detecting human alpha-fetoprotein is characterized in that: the monoclonal antibody Ab1 or/and Ab3 is a capture antibody, the monoclonal antibody Ab2 of claim 1 is a detection antibody;

wherein, the amino acid sequence of the heavy chain variable region of the monoclonal antibody Ab1 is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8;

3. The monoclonal antibody of claim 1 in preparing kit or reagent for early diagnosis of liver cancer.

4. Use of the monoclonal antibody of claim 1 for the preparation of a kit or reagent for the detection of human alpha-fetoprotein.